Snapshot of an oxygen intermediate in the catalytic reaction of cytochrome oxidase.

Cytochrome oxidase (CO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CO. It is assigned to the P-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme iron atom is in a ferryl (Fe = O) configuration, and heme and Cu are oxidized while Cu is reduced. A Helix-X segment is poised in an open conformational state; the heme farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.